Water-soluble tissue infiltrating and embedding compositions

ABSTRACT

Water-soluble tissue infiltrating and embedding compositions facilitating rapid preparation of tissue sections biopsic study comprising 50 to 60 percent of polyethylene glycol having a molecular weight of about 3700 and somewhat less than 50 percent of a mixture of nonionic surface active agents having varying consistency ranging from solid to semisolid and liquid consistencies together with small amounts of water soluble and water-insoluble waxes, whereby the hardness of the polyethylene glycol is reduced and hygroscopicity maintains substantially constant during seasonal variations of ambient temperature and humidity.

United States Patent Inventor Philip Schain 126 Silver Lake Road, Staten Island, N.Y. 10301 Appl. No. 871,754

Filed Sept. 4, 1969 Patented Nov. 30, i971 Continuation of application Ser. No. 555,969, June 8, 1966, now abandoned. This application Sept. 4, I969, Ser. No. 871,754

WATER-SOLUBLE TISSUE INFILTRATING AND [56] References Cited UNITED STATES PATENTS 3,257,279 6/1966 Schain 424/3 OTHER REFERENCES Carbowax, booklet. Union Carbide Chem. Co. N Y N58 Primary Examiner-Albert T. Meyers Assistant Examiner-A. P. Fagelson Attorney-Howard E. Thompson, .lr.

ABSTRACT: Water-soluble tissue infiltrating and embedding compositions facilitating rapid preparation of tissue sections biopsic study comprising 50 to 60 percent of polyethylene glycol having a molecular weight of about 3700 and somewhat less than 50 percent of a mixture of nonionic surface active agents having varying consistency ranging from solid to semisolid and liquid consistencies together with small amounts of water soluble and water-insoluble waxes, whereby the hardness of the polyethylene glycol is reduced and hygroscopicity maintains substantially constant during seasonal variations of ambient temperature and humidity.

WATER-SOLUBLE TISSUE INFILTRATING AND EMBEDDING COMPOSITIONS This application is a continuation of application, Ser. No. 555,969, filed June 8, 1966, now abandoned.

This invention relates to chemical compositions adapted and intended primarily for use as water-soluble tissue infiltrating and embedding compositions facilitating rapid preparation of tissue sections for biopsic study. More particularly the invention relates to water-soluble tissue infiltrating and embedding compositions which melt below about 50 C., and in the molten state can readily penetrate both acqueous and fat containing tissues, and which when set provide a firm, slightly elastic, and slightly self-adhering consistency facilitating the cutting of thin tissue sections for biopsic examination. Still more particularly the invention relates to compositions of the class described having enhanced nonhygroscopic properties permitting their use with essentially uniform results in spite of wide variations in temperature and humidity conditions.

In my pending U.S. application Ser. No. 213,623 filed July 3], 1962 now U.S. Pat. No. 3,257,279 issued June 21, 1966 water-soluble tissue infiltrating and embedding compositions are disclosed which consist predominantly of a mixture of different alkyl phenol ethylene oxide condensates, i.e. wherein the mols of ethylene oxide per mol of nonylphenol differ substantially, together with minor amounts of waxes and other components. For example, a preferred formulation disclosed in said application contains 97 percent by weight of such alkylphenol ethylene oxide condensates. While it was considered that such preferred formulation was essentially nonhygroscopic it has been found that under the conditions of high temperature and humidity frequently encountered in the summertime the composition in fact can be sufficiently hygroscopic so that the desired combination of firmness, elasticity, and self-adhesion is partially lost or impaired, with the result that satisfactory biopsic specimens cannot readily be prepared under such conditions of high temperature and humidity. Furthermore in attempting to modify the compositions within the teachings of said prior application it is found to be rather difficult to provide in one composition satisfactory properties and performance under conditions of high temperature and humidity and equally satisfactory performance under lower conditions of temperature and humidity which are more frequently encountered. It would appear, therefore, that following the teachings of said pending application the best way to provide optimum performance under different temperature and humidity conditions would be to provide two or more different compositions from which to select the proper composition based upon the temperature and humidity at the time particular biopsic studies are to be conducted.

lt has now been discovered in accordance with the present invention that the problems above mentioned can be overcome by switching to a totally modified composition containing a preponderant amount i.e. 50 to 60 percent of polyethylene glycol having a molecular weight of about 3700 and somewhat less than 50 percent of a mixture of nonionic surface action agents and small amounts of water-soluble and water-insoluble waxes. ln this new type composition the polyethylene glycol, which by itself is much too hard for use as a tissue embedding agent, appears to provide a lowering of hygroscopicity which is not ofi'set by the more hygroscopic nonionic components; and the new compositions have been found to perform satisfactorily under all conditions of varied temperature and humidity normally encountered.

The several components of the new compositions are presented in the following tabulation together with the preferred weight percent and permissible range for each:

2. Water-soluble nonionic C,,-C,,, alkyl phenoloxide of solid nonyl phenol ethylene oxide condensate containing about 40 mols of ethylene oxide per mol of nonyl phenol Water-soluble nonionic polynlkylene glycol ether of semisolid consistency; butoxy ether of mixed (ethylene-propylene) polyalkylene glycol having a molecular weight of about 3000-3500, a cloud point of in a l% aqueous solution. a specific gravity of L034 (70/20" C.). a solidification temperature of about 36-46 C.. a surface tension of 39 dynes/cm. at 25 C., and weighing 8.72

lbs/gal. at 50 C. (Tergitol XH) Water-soluble polyethylene glycol of solid consistency having a molecular weight of l5,000 to 5. Fat-soluble water-insoluble) nonionic C C alkyl phenol ethylene oxide condensate of liquid consistency, nonyl phenol ethylene oxide condensate containing 4 to 7 mols of ethylene oxide per mol of nonyl phenol 6. Paraffin (watehinsoluble) Also as an optional component a paraffin-miscible natural wax such as carnauba wax, beeswax, or mixtures thereof While items two and five above mention specifically nonyl phenol ethylene oxide condensates it is to be understood that the closely related octyl phenol and decyl phenol ethylene oxide condensates have quite similar properties and can readily be used. The nonyl phenol condensates are preferred due to their low cost and general availability.

The composition is prepared by melting and mixing together at about 60 C. the water-soluble components one, two three and four, separately mixing together at about 60 C. components five and six (and seven if present), and adding the later to the former with continued heating until the entire composition is clear. Upon cooling, the solid composition should have a melting temperature of 43 to 50 C.

The composition containing the preferred amounts of the various components in the foregoing tabulation is a general purpose composition which when melted readily penetrates the mixed aqueous and fatty tissues normally encountered in biopsic specimens. For special purposes the composition can be modified to vary the hardness, elasticity, degree of hygroscopicity, and penetrability into aqueous and fatty tissue by varying amounts of the components within the limits above described. As a guide to such modification of the composition, it should be noted that:

a. Hardness is increased by using larger quantities of components one and four, including some carnauba wax as an optional component, and correspondingly decreasing component 3.

b. Elasticity is increased by increasing the amount of component three, while correspondingly decreasing the amount of components one and four, and/or utilizing beeswax in place of at least part of the paraffin.

c. Hygroscopicity can be decreased by taking the steps outlined in a" above as well as by increasing the mount of parafi'm and paraffin miscible components.

d. Penetrability into aqueous tissue is increased by an increase in component two with a corresponding decrease in components five and six.

e. Penetrability into fatty tissue is increased by an increase in component 5, as well as increases in components three and six with a corresponding decrease in component two.

As modified by any of the procedures above mentioned the composition should melt at about 4350 C.

ln utilizing the new compositions for the preparation of tissue specimens for biopsic examination the following procedure is followed:

A tissue sample is first immersed for 2 to 3 hours in polyethylene glycol having a molecular weight of 950-1050 (Carbowax 1000) at a temperature of about 55 to 60 C., followed by immersion for 20 to 30 minutes in a 55 to 60 C. melt of the above described composition. After this 20 to 'done under vacuum the time can be further reduced to less than 2 hours.

The blocked tissue specimen can then be cut into thin, uniform sections which have a firmness and flexibility which permits the cut sections, if desired, to adhere to each other to form long ribbons, as is important in serial studies of tissue. Furthermore, when the cut sections are placed in water, the tissue hardening composition is rapidly dissolved, and within 1 minute, and generally within to 30 seconds, the tissue specimens can be directly stained by conventional water-soluble stains.

Various changes and modifications in the composition herein disclosed will occur to those skilled in he art, and to the extent that such changes and modifications are embraced by the appended claims, it is to be understood that they constitute part of the present invention.

I claim:

1. A water-soluble tissue infiltrating and embedding composition facilitating rapid preparation of tissue sections for biopsic study, said composition consisting essentially of:

a. 50 to 60 percent by weight of water-soluble polyethylene glycol having a molecular weight of about 3700,

b. to percent of a water-soluble C to C alkyl phenol ethylene oxide condensate of solid consistency,

c. 13 to 23 percent of a water-soluble nonionic polyalkylene glycol ether of semisolid consistency comprising the butoxy ether of mixed (ethylene-propylene) polyalkylene glycol having a molecular weight of about 3,000 to 3.500, a cloud point of in a 1 percent aqueous solution, a specific gravity of 1.034 (70/20 C.), a solidification temperature of about 36-46 C., a surface tension of 39 dynes cm. at 25 C., and weighting 8.72 lbs/gal. at 50 C.

d. l to 5 percent of water-soluble polyethylene glycol having a molecular weight of 15,000 to 20,000,

e. 0.2 to 3.6 percent of fat-soluble, water-insoluble nonionic C to C alkyl phenol ethylene oxide condensate of liquid consistency,

f. 0.3 to 1.8 percent of parafiin, and

g. 0 to 1.8 percent of a paraffin miscible natural wax;

said composition in the molten state being a clear solution capable of penetrating both aqueous and fatty tissue, and setting at about 43 to 50 C. to a water-soluble waxlike solid, and optimum balance of the components of said composition imparting to embedded tissue sections a firmness, flexibility and self-adhesion such that a plurality of cut sections will adhere to each other in the form of long ribbons.

2. The water-soluble tissue infiltrating and embedding composition of claim 1 wherein component (b) is a nonyl phenol ethylene oxide condensate containing about 40 mols of ethylene oxide per mol of nonyl phenol, component (e) is a nonyl phenol ethylene oxide condensate having about 4 to 7 mols of ethylene oxide per mol of nonyl phenol, and component (g) is selected from the group consisting of carnauba wax, beeswax, and mixtures thereof. 

2. The water-soluble tissue infiltrating and embedding composition of claim 1 wherein component (b) is a nonyl phenol ethylene oxide condensate containing about 40 mols of ethylene oxide per mol of nonyl phenol, component (e) is a nonyl phenol ethylene oxide condensate having about 4 to 7 mols of ethylene oxide per mol of nonyl phenol, and component (g) is selected from the group consisting of carnauba wax, beeswax, and mixtures thereof. 